Uveal melanoma (UM), the most common intraocular malignancy in adults, is uniformly refractory to all available systemic chemotherapies, and, as a result, is universally lethal when metastatic, creating an unmet need for novel, effective, targeted therapies for this orphan disease. Somatic activating mutations in G(alpha)q and G(alpha)11, present in a mutually exclusive pattern in ~80% of UMs, activate the PKC pathway and function as bona fide oncogenic drivers. As the O'Malley lab had previously reported that steroid receptor coactivator-3 (SRC-3), a transcriptional coactivator with potent growth-promoting activity, is post-translationally stabilized via PKC-mediated phosphorylation, we decided to build upon our experience in SRC-3 studies and examine the role of SRC-3 in UM. Our preliminary studies suggest that G(alpha)-induced oncogenic signaling is mediated by the protein kinase C (PKC)alpha/protein kinase D (PKD) pathway, and leads to stabilization of SRC-3, which then co-localizes on chromatin and co-operates with microphthalmia-associated transcription factor (MITF), a critical transcription factor for melanocytes, to drive oncogenic signaling. G(alpha)q-mutant UM cells are exquisitely dependent on SRC-3 for proliferation/survival in vitro and in vivo. Small molecule inhibitors (SMIs) of PKC cause depletion of SRC-3 protein and exert anticancer activity in G(alpha)q-mutant UM cells, while restoration of SRC-3 expression rescues the viability of UM cells that have been treated with G(alpha)q/PKC inhibition. Based on these recent findings, our core hypothesis is that mutant G(alpha) proteins trigger PKC-mediated intracellular oncogenic signaling that stabilizes SRC-3, which then functions as a coactivator for the transcription factor MITF. Moreover, pharmacological inhibition of SRC-3/MITF will exert potent anticancer activity, thus providing an innovative therapeutic opportunity for UM patients. Our proposal provides the roadmap for the development of such innovative therapies for this highly lethal orphan disease. Our aims are to elucidate the role of SRC-3 in UM pathophysiology and resistance to systemic therapy (in particular PKC inhibitors), and to establish it as a therapeutic target in UM, using a large panel of UM cell lines, primary cultures of UM cells and patient biopsies; examine the cooperation between SRC-3 and MITF, define their transcriptional target genes, determine how these transcriptional targets are regulated by MITF and SRC- 3, and dissect their functional significance in UM; define the activity of newly identified SRC-3/MITF pathway SMIs as a novel therapeutic approach for UM, in particular for disease resistant to targeted therapies using in vitro and in mouse UM models (both as monotherapies and in combination regimens). Our goal is to develop novel targeted therapeutics for UM, with particular emphasis on rationally-designed combinatorial approaches, in order to overcome resistance to treatment. Collectively, our research strategy will enhance our understanding of UM pathophysiology and provide novel targets and therapeutic agents.